1. Field of the Invention
The invention relates to the production of ethanol and more particularly, to a method for the production of ethanol in high yields from cellobiose fermenting yeast Brettanomyces custersii in a simultaneous saccharification and fermentation process employing cellulosic materials as a substrate.
2. Description of the Prior Art
In the past, the production of ethanol has been procured by a procedure comprising the steps of reacting cellulase upon cellulose as the substrate to enzymatically saccharify the cellulose to glucose, and then separately causing the resultant glucose to be reacted upon by an ethanol-producing microorganism to produce ethanol. However, using this conventional method, the conversion of cellulose to glucose by a cellulase is low and, as a consequence, large amounts of unconverted cellulosic residue are obtained. Accordingly, low yields of ethanol are obtained by subjecting the saccharified liquid to fermentation.
Nevertheless, ethanol is of great interest to a wide variety of industries due to past and potential future energy crisis situations, and the conversion of these cellulose materials to useful fuels such as ethanol is of special interest.
Cellulose and hemicellulose are the two most abundant and renewable raw organic compounds in the world and together they compose about 70 percent of the entire world's plant biomass on a dry weight basis. These raw materials are widely available in the waste from agricultural, forest, vegetable, and food process sources and the efficient conversion of these wastes to useful products such as ethanol, would help reduce disposal problems as well as provide an abundant and cheap source of fuel. Other cellulose containing biomass can be grown specifically to provide a feedstock for ethanol manufacture and to support wide scale ethanol production for use as a fuel.
More specifically, plant biomass generally contains from 40-50 percent cellulose and 30-40 percent hemicellulose, and a balance of lignin. If a process for converting the bulk of the cellulose and hemicellulose to ethanol in high yields could be devised, this process could provide an almost unlimited supply of liquid fuel for transportation uses.
Cellulose is readily broken down to its glucose and cellobiose hydrolysate products by acid hydrolysis or enzymatic hydrolysis treatment. While glucose is readily fermentable by many microorganisms to ethanol, cellobiose has proven difficult, at best, to convert to ethanol. Even then, it is convertible to ethanol only in very low yields or low concentrations. (R. Dekker, Biotechnology Letters, Volume 4, No. 7, Pages 411-416, 1982; R. Maleszka, et al., Biotechnology Letters, Volume 4, No. 2, pp. 133-136, 1982).
Hemicellulose is likewise readily and easily converted to its various hydrolysate products by mild acid hydrolysis or enzymatic hydrolysis treatment and the resultant products include various pentoses (xylose and arabinose being the main derivatives), hexoses (mannose and galactose), and sugar acids. By far, D-xylose is the major sugar in hemicellulose hydrolysate and constitutes approximately 60-80 percent of the total hydrolysates produced therefrom.
A variety of processes which use different yeasts to ferment xylose to ethanol have been investigated and disclosed in the literature. A prime motivating force behind these investigations is that the fermentation of 5-carbon sugars derived from hemicellulose is extremely important in order to fully utilize biomass material in producing ethanol. Examples of such prior art techniques include U.S. Pat. Nos. 4,511,656, 4,368,268, 4,359,534, and 4,477,569. However, these processes do not convert D-xylose to ethanol in sufficient yields and at sufficiently high rates to be efficient and cost effective.
U.S. Pat. No. 4,385,117 pertains to a process for continuously producing ethanol such that a substrate can be added to a fermentation and the ethanol can be removed therefrom during a fermentation comprising, subjecting an aqueous nutrient medium containing the substrate at a substrate concentration in the fermentation medium greater than one percent (w/v) wherein the substrate is starch, pectin, monosaccharides and disaccharides, under anaerobic and thermophilic conditions to the fermentation action of a derivative of the microorganism Thermoanaerobacter ethanolicus; however, this patent is directed to a thermophilic rather than a mesophilic microorganism which is reported on substrates greater than one percent, and where it is apparent that a sharp decrease in ethanol tolerance on starch concentrations above one percent exist, and this of course is a very low substrate loading in a saccharification and fermentation process.
U.S. Pat. No. 4,464,471 is directed to a recombinant DNA plasmid comprising a cloning vector having covalently bound thereto a DNA insert coding for the production of beta-glucosidase wherein the DNA insert coding for the production of beta-glucosidase is isolated from Esherichia adecarboxylata; but this patent is directed to a genetically engineered microorganism via plasmid coding, and this is not a natural organism and it has not been demonstrated in simultaneous saccharification fermentation processes.
U.S. Pat. No. 4,472,501 pertains to a process for producing ethanol comprising culturing a microorganism from the group of microorganisms having the identifying characteristics of Kluyveromyces cellobiovorus NRRL Y-12509 and a microorganism having the identifying characteristics of Kloeckera apiculata NRRL Y-12510 and which is capable of producing ethanol and assimilating at least one carbon source selected from xylose and cellobiose, in a medium containing an assimilable source of at least one of said xylose and cellobiose until a recoverable amount of ethanol is produced in the culture liquor and thereafter recovering said ethanol therefrom; however, the microorganisms are different from those of the invention and said microorganisms are compared in their performance to a glucose fermentor (Saccharomyces cerevisiae) albeit one of their organisms demonstrates a low ethanol yield on crystalline cellulose and the others were fermented on glucose, xylose and/or cellobiose.
In U.S. Pat. No. 4,840,903, there is disclosed a process for producing ethanol from plant biomass by forming a substrate from said biomass, wherein the substrate includes hydrolysates of cellulose and hemicellulose, after which a species of the fungus Paecilomyces, which has the ability to ferment both cellobiose and xylose to ethanol, is then selected and isolated. The substrate is inoculated with said fungus and the inoculated substrate is fermented under conditions favorable for cell viability and conversion of hydrolysates to ethanol, and the ethanol is recovered from the fermented substrate; however, the conversion of cellobiose and xylose to ethanol is by the mechanism of a fungus, and not by a yeast, and the yields and rates of ethanol are not sufficient for the simultaneous saccharification process to be economically feasible.